Substrate-dependent modulation of the leukotriene A4 hydrolase aminopeptidase activity and effect in a murine model of acute lung inflammation

The aminopeptidase activity (AP) of the leukotriene A4 hydrolase (LTA4H) enzyme has emerged as a therapeutic target to modulate host immunity. Initial reports focused on the benefits of augmenting the LTA4H AP activity and clearing its putative pro-inflammatory substrate Pro-Gly-Pro (PGP). However, recent reports have introduced substantial complexity disconnecting the LTA4H modulator 4-methoxydiphenylmethane (4MDM) from PGP as follows: (1) 4MDM inhibits PGP hydrolysis and subsequently inhibition of LTA4H AP activity, and (2) 4MDM activates the same enzyme target in the presence of alternative substrates. Differential modulation of LTA4H by 4MDM was probed in a murine model of acute lung inflammation, which showed that 4MDM modulates the host neutrophilic response independent of clearing PGP. X-ray crystallography showed that 4MDM and PGP bind at the zinc binding pocket and no allosteric binding was observed. We then determined that 4MDM modulation is not dependent on the allosteric binding of the ligand, but on the N-terminal side chain of the peptide. In conclusion, our study revealed that a peptidase therapeutic target can interact with its substrate and ligand in complex biochemical mechanisms. This raises an important consideration when ligands are designed to explain some of the unpredictable outcomes observed in therapeutic discovery targeting LTA4H.

by digesting and clearing PGP since accumulation of PGP activates the CXCR2 receptors on neutrophils 5,[16][17][18] . Therefore, bi-functional EH and AP activities undergird the intricate pro-and anti-inflammatory regulation, respectively, exerted by LTA 4 H.
Snelgrove reported that lipopolysaccharide (LPS) exposure causes bioproduction of PGP and acute lung injury 5 . Unlike cigarette smoke exposure, LPS exposure does not suppress endogenous LTA 4 H AP activity, and therefore, active LTA 4 H AP clears PGP after exposure to LPS in murine lung 5 . Numao reports additional observations, which show that PGP does not induce inflammatory responses at micromolar concentrations found in the murine air-pouch model of inflammation 19 . This report on PGP is puzzling since several independently corroborating reports demonstrate that the in vivo levels of PGP correlate with host inflammatory responses and outcomes 5,6,20 . These contradicting reports also suggest that cigarette smoke causes lung injury by an accumulation of acrolein and acidification of the airways, which result in an accumulation of PGP in the lungs via suppression of LTA 4 H AP activity 6,8,21 . In murine models of lung injury, reducing PGP levels in the lung by 4-methoxydiphenylmethane (4MDM) correlates with the resolution of neutrophilic inflammation and other beneficial therapeutic effects 6,20 . 4MDM selectively augments the LTA 4 H AP activity and does not affect the EH activity 6,20 . 4MDM has minimal off-targeting effects reported by our group as demonstrated in an LTA 4 H knockout murine model 6 . We have demonstrated that treatment with 4MDM restores the LTA 4 H AP activity and prevents PGP accumulation by cigarette smoke exposure 6 . To clarify the contradicting reports on PGP, we set forth to conduct studies using 4MDM as a pharmaceutical tool to interrogate the biochemistry of the LTA 4 H AP activity.
First, we characterized the effects of selectively augmenting LTA 4 H AP activity on pulmonary inflammation induced by LPS. We have taken advantage of this unique aspect with the LPS murine model to determine the effect of selectively augmenting LTA 4 H AP activity with 4MDM in a PGP-independent model for pulmonary inflammation with 4MDM. Since PGP interaction with LTA 4 H is not completely characterized biochemically, our study is mainly focused on the AP activity of LTA 4 H in the presence of 4MDM. In addition to PGP, dynorphin and enkephalin are peptide-based substrates for the LTA 4 H AP activity [9][10][11][12] . As reported by Orning and co-workers, LTA 4 H hydrolyzes tripeptides containing an N-terminal arginine more efficiently than other peptide substrates such as dipeptides, tetrapeptides, pentapeptides, and tripeptides with non-arginine N-termini 22 . Therefore, LTA 4 H can be considered an arginine N-aminotripeptidase 22 . In order to gain more insight into the substrate-specific properties of LTA 4 H AP activity, we determined the effect of 4MDM on the kinetic mechanisms for hydrolysis of PGP, Ala-pNA, Arg-pNA, and Pro-pNA. Lastly, we reported the first X-ray crystal structures of LTA 4 H in complex with 4MDM and LTA 4 H bound to 4MDM and N-(4-oxo-4-pyrrolidinyl-butanoyl)-proline (OPB-Pro), a non-hydrolyzable analogue of PGP.

Regulation of LTA 4 H bifunctionality in the murine model of acute lung inflammation and injury induced by intra-nasal LPS.
The experimental design of the murine model is shown in Fig. 1A. Mice were treated daily from Days 0 to 4 with intranasal (IN) 4MDM after being exposed to IN LPS on Day 0. LTA 4 H AP activity was significantly elevated after five days of 4MDM treatment over that of the vehicle (Fig. 1B). Levels  www.nature.com/scientificreports/ of LTB 4 in the bronchoalveolar lavage fluid (BALF) were comparable between the 4MDM-and vehicle-treated animals (Fig. 1C). These studies indicated that 4MDM treatment selectively enhanced the LTA 4 H AP activity without affecting the EH activity in the murine model of LPS-induced acute lung injury. PGP concentration was assessed in the whole lung BALF collected from two treatment cohorts after LPS exposure (vehicle vs. 4MDM) on Days 1 and 5. While the PGP levels in the BALF were above the level of detection, they were well below the lower limit of quantification except in the BALF samples from the cohort treated with 4MDM for five days. In this model, the concentration of PGP was at a level where its biological contribution was believed to be trivial (Tables S1 and S2). 4MDM treatment consistently reduced the number of CD45 + leukocytes and CD45 + CD11b + Ly6G + neutrophils in the LPS-exposed lungs on Days 1 and 5 ( Fig. 2A,B), while LTA 4 H activity in BALF was significantly augmented on Day 5 (Fig. 1B). LTA 4 H activity measured in the BALF may not fully reflect that of activity in lung tissue. The severity of acute lung injury was assessed by wet-to-dry lung weight ratio and Sireq Flexivent pressure-volume loop. 4MDM treatment maintained the lung to be more compliant by the premortem pressure-volume loop, consistent with less water content and less severe acute lung injury (Fig. 2C,D). Representative H&E staining shows significantly fewer leukocytes infiltrating the peri-bronchial areas (arrow) with 4MDM treatment than with vehicle alone (Fig. 2E).
Enzyme kinetics of LTA 4 H for the hydrolysis of PGP, Arg-pNA, Ala-pNA, and Pro-pNA in the presence of 4MDM. The reaction velocity plots of PGP hydrolysis were determined at escalating concentrations of 4MDM (Fig. 3). The data is consistent with substrate-induced inhibition at increasing substrate concentrations. Our data suggested that a substrate-enzyme-substrate (SES) complex formed, which would involve the addition of a second PGP molecule to the LTA 4 H-PGP complex 23,24 . 4MDM accentuated PGP-induced inhibition in a dose-dependent manner.
Enzyme kinetics studies were performed to elucidate the kinetic mechanism of LTA 4 H-mediated hydrolysis of Arg-pNA, Ala-pNA, and Pro-pNA at escalating concentrations of 4MDM (Figs. 4, 5, 6). 4MDM induced hyperbolic predominantly specific inhibition in the presence of Arg-pNA, hyperbolic mixed predominantly catalytic activation with Ala-pNA, and hyperbolic catalytic activation with Pro-pNA.
These studies revealed the effect of 4MDM on LTA 4 H kinetics by perturbing the equilibrium coupling constant α and the enzyme kinetic catalytic constant β. Decreasing values for α signify increasing stabilization of the enzyme-modifier-substrate (EXS) complex where X is 4MDM, E is LTA 4 H, and S is amino acid-pNA substrate (Fig. 4). Therefore, 4MDM is likely an unsuitable activator for potential R-X-X substrates, because it inhibits Arg-pNA binding competitively by stabilizing the formation of the EX complex. Given the high α value for hydrolysis of Arg-pNA in the presence of 4MDM, 4MDM and Arg-pNA cannot simultaneously bind to LTA 4 H, presumably due to the large size of the Arg side chain. However, 4MDM predominantly activates Ala-pNA hydrolysis by increasing catalytic turnover (β > 1) for the EXS complex 25 . In contrast to Arg-and Ala-pNA mechanisms, 4MDM "uncompetitively" activates Pro-pNA hydrolysis where the equilibrium coupling constant α equals the kinetic www.nature.com/scientificreports/ catalytic constant β (α = β). The catalytic specificities and kinetic parameters for hyperbolic enzyme modifications of Arg-, Ala-, Pro-pNA are shown in Tables 1 and 2 23 . These studies suggested that for activation, 4MDM does not universally activate the LTA 4 H enzyme and its ability to activate or inhibit hydrolysis highly depends on the nature of the substrate. Notably, 4MDM affects the ES complex by contributing to partial predominantly catalytic activation (Ala-pNA) or exclusive catalytic activation (Pro-pNA).

Crystal structure of LTA 4 H in complex with 4MDM and 4MDM:OPB-Pro. Human recombinant
LTA 4 H was co-crystallized with 4MDM ( Fig. 7A) and 4MDM:OPB-Pro (Fig. 7B), where OPB-Pro is a nonhydrolyzable analog of PGP. The full-length structure of LTA 4 H in complex with 4MDM was refined at 2.9 Å to R-work of 18% and R-free of 21%. The LTA 4 H-4MDM complex crystallized in space group P3 2 , with three molecules in the asymmetric unit. As shown in the M1 metallopeptidase HEXXH-(X) 18    . The kinetic scheme of non-essential mixed type activation and rate constants (Baici, 2015). The k 1 , k 2 , k 3 , k 4 , and k 5 are rate constants, and the unit with k 2 , k 3 , and k 5 in s −1 , and the unit of k 1 and k 4 in M −1 s −1 . The corresponding dissociation constants K S and K X , in M, equal k 2 /k 1 and k 5 /k 4 , respectively. The Michaelis-Menten constant K m equals (k 2 + k 3 )/k 1 with units of M. The catalytic constant k cat (k 3 ) is in s −1 . α and β are dimensionless positive coefficients. K Sp and K Ca refer to the specific (competitive) and catalytic (uncompetitive) modifications, respectively. The catalytic efficiency k cat /K m of the enzyme is used for comparing the relative rates of enzyme activity acting on the substrate with or without modifiers.  27 . The polar side chain of Q136 was oriented toward the aminopeptidase active site in the LTA 4 H:4MDM complex, whereas it was oriented toward the hydrophobic cavity in the LTA 4 H:OPB-Pro structure (PDB ID: 4MS6) 27 . Hydrogen bonding interactions were present between the O ε1 atom of Q136 and the Cγ atom of OPB-Pro. In the published LTA 4 H:ARM1:OPB-Pro structure (PDB ID: 4MKT) 27 , two rotamers were observed for the side chain of Q136, while the side chain of Q136 in the LTA 4 H:4MDM:OPB-Pro structure showed only one orientation, where the side chain orients toward the catalytic Zn 2+ cation. Also, the crystal structure of LTA 4 H:4MDM showed that Q136 was oriented towards the Zn 2+ cation, which has the same orientation shown in the structure of LTA 4 H:4-OMe-ARM1 complex (PDB ID: 6O5H) 25 . This additional structural information provided by the LTA 4 H:4MDM and LTA 4 H:4MDM:OPB-Pro complexes indicates that Q136 could have a role in tripeptide substrate recognition and catalytic turnover by maintaining rotational freedom 28 . In both 4MDM bound structures,   (Fig. S3). We analyzed that the LTA 4 H:4MDM:OPB-Pro complex created a lower dielectric environment, which induced the release of these non-catalytic water molecules. The loss of water molecules could afford more favorable binding entropies for nonpolar ligand binding, provided steric clashes are avoided.

Discussion
Numerous human pathologies correlate with dysfunctional LTA 4 H activities that result in an accumulation of LTB 4 and PGP 5,8,14 . Research studies suggest that PGP is a pro-inflammatory matrikine that is produced during host tissue injury 18 . While host proinflammatory responses seem to be strongly associated with elevated levels of PGP, a recent study questions the biological activity of PGP. Numao and co-workers suggest that the antiinflammatory LTA 4 H AP activity may result from clearance of substrates other than PGP, implying that even at high levels, PGP might be biologically less relevant to inflammatory signaling 19 . Therefore, we employed a murine model of LPS-induced pulmonary neutrophilic inflammation to ascertain the biology of LTA 4 H AP activity independent of PGP. Previous studies have shown that IN LPS induces acute neutrophilic inflammation in lungs while simultaneously inducing the LTA 4 H enzyme levels 29,30 . Past studies showed that PGP levels declined rapidly within 24 h due to endogenous LTA 4 H AP activity after exposure to LPS 5 . Numao also reported that the levels of PGP observed in the murine lung exposed to LPS did not cause significant neutrophilic inflammation 19 . Therefore, we concluded that the murine pulmonary inflammation model induced by LPS was a suitable model to ascertain if at least a part of the LTA 4 H AP biology is unrelated to PGP. In this model, perturbation of the LTA 4 H AP activity by 4MDM will provide additional corroboration. We construed that if the LTA 4 H AP-mediated hydrolysis of PGP is a significant anti-inflammatory pathway, 4MDM should not alter LPS-induced neutrophilic inflammation because PGP, the putative target of the LTA 4 H AP activity, is cleared rapidly after LPS exposure.
Our study demonstrated that LTA 4 H AP activity was selectively augmented by 4MDM when Ala-pNA or Pro-pNA were used as reporters, which corresponded to significantly reduced airway neutrophilia caused by IN LPS throughout the 5-day observation period. PGP levels in BALF were low or undetectable, suggesting that the biological effects of the augmented LTA 4 H AP activity is independent of PGP. Beneficial effects were also independent of LTB 4 levels. These results led us to conclude that augmentation of the LTA 4 H AP activity brings broader biological effects beyond the clearance of PGP. These observations bring forth a speculation that the LTA 4 H AP activity is not exclusive to the clearance of PGP, and therefore raises the need for detailed enzymatic studies on the LTA 4 H AP activity with substrates other than PGP. www.nature.com/scientificreports/ Findings from our studies reveal several previously underappreciated biochemical mechanisms. First, PGP interacts with LTA 4 H AP by a substrate-induced inhibition mechanism. Under this mechanism, increasing concentrations of PGP would negatively feedback the LTA 4 H AP activity. Besides accelerating PGP accumulation, the substrate-induced inhibition may cause persistent inflammation by inhibiting LTA 4 H AP activity, assuming robust LTA 4 H AP activity is required to maintain the anti-inflammatory functions of LTA 4 H. Second, the enzyme kinetics significantly diverged depending on substrates. Arg-pNA, Ala-pNA, and Pro-pNA interact with the LTA 4 H AP substrate site under three distinctive enzymatic mechanisms in the presence of 4MDM. While 4MDM primarily inhibited hydrolysis of Arg-pNA, 4MDM enhanced the hydrolysis of Ala-pNA and Pro-pNA. Of these three substrates, the enzyme kinetics of Pro-pNA was most intriguing as it followed a rarely observed hyperbolic catalytic activation (HCaA) mechanism, which is analogous to an "uncompetitive inhibition" mechanism where the modulator has higher affinity for the enzyme-substrate complex than to the enzyme. Surprisingly, PGP was further inhibited by the presence of 4MDM, which showed us that the structure of the N-terminus of the reporter group is insufficient to report the enzyme kinetics for corresponding peptides. Regardless, we speculate that the effect of PGP hydrolysis may not be relevant for our preclinical model.
Comparisons with previously published X-ray crystal structures of LTA 4 H:R-X-X tripeptides complexes and the LTA 4 H:4MDM complex can help explain our enzyme kinetics data from a structural perspective. X-ray crystal structures of LTA 4 H complexes demonstrate that RSR and RAR tripeptides bind the GXMEN motif in an extended β strand conformation 31 . The structures also reveal that Asp375, a key residue for argininyl-tripeptide hydrolysis, forms hydrogen bonding interactions with the guanidinium group of arginine to stabilize the N-terminal arginine residue and position the tripeptide for hydrolysis within the AP active site 28,31 . In addition, Arg563 and Lys565 are key residues for tripeptidase function as they cooperate with each other for strong alignment of Active site Zn 2+ and coordinating His residues are indicated. The direction of the Gln-136 side chain points toward the hydrophilic binding site with respect to water. The direction of the methoxy group in 4MDM is rotated in the tri-complex. Background residues were deleted to enhance the visibility of the binding site except for residues from Ser-133 to Arg-141, from Asn-291 to Ser-300, and from Thr-310 to Val-376 are shown in line rendering.  (Fig. S4), which agrees with our enzyme kinetics data that 4MDM inhibits N-terminal arginine substrate by a specific inhibition mechanism. The overlays of these X-ray crystal structures with the LTA 4 H:4MDM crystal structure demonstrates that 4MDM modulates peptidase activity through its influence on the hydrophobic interactions and rotational freedom of Q136. Also, the structural data suggest that nonpolar ligands are likely necessary for enhancing the peptidase activity due to more favorable binding entropies in the active site. Hence hydrophobic interactions and steric considerations are the primary factors influencing what modified kinetics may be possible. Hydrolysis of peptides with an N-terminus containing a smaller hydrophobic side chains are more likely activated by 4MDM, whereas larger or more polar side chains would likely be inhibited by 4MDM.
In conclusion, our studies demonstrate biochemical mechanisms of the LTA 4 H AP activity in details that were not previously attempted. Since the studies by Numao and, now, our studies are raising a possibility of more complex biological activities exerted by the LTA 4 H protein and its substrates 19 , a simple assessment of IC 50 or AC 50 to screen therapeutic molecules seems inadequate. Our data strongly suggest LTA 4 H AP activity, as modulated by 4MDM, is biologically relevant for anti-inflammatory responses. This response, however, appears independent of its action on PGP. These observations merit attention, because most previous methods seem incomplete and premature to determine even the simple therapeutic effects based on biochemical enzymatic activities. Development of any compounds targeting LTA 4 H will require complete enzymatic characterization as we have done in our recently published work 25 . Moreover, a single therapeutic agent such as 4MDM may experience substratedependent mechanisms, leading to highly divergent interactions (such as inhibition or activation) with LTA 4 H. This is clearly an underappreciated aspect of LTA 4 H biology. The potentially opposite therapeutic effects a single compound can generate in vivo complicates the search for therapies targeting LTA 4 H. More careful assessment and characterization of LTA 4 H enzymatic activities are much desired to further inform strategies for developing therapeutics for this important target. We recommend reexamining the mechanistic studies on inflammatory responses with regards to the complex activities of LTA 4 H and to base the discovery of new therapeutic agents on the distinct modification mechanisms of the enzyme. Fine Chemical, LLC) and 4MDM. Crystals were obtained from drops containing 60-90 mM magnesium formate dihydrate and 20-25% PEG3350 at 22 °C. Both crystal diffraction data were collected at 100 K in-house at the WRAIR X-ray Diffraction Facility using a Bruker Microstar rotating anode X-ray generator with a Pt 135 CCD detector. For data collection, crystals were cryoprotected in mother liquor with the addition of 25% ethylene glycol and frozen in a N 2 gas stream. The data was reduced with the Proteum software from Bruker, and the structure was determined by molecular replacement using the program Phaser within the PHENIX suite. The LTA 4 H structure (PDB ID: 4MS6) was used as a search model for phasing following the removal of water and ligands. 4MDM and Zn 2+ molecules were built within Fo-Fc density using coot with refinement in PHENIX. www.nature.com/scientificreports/ tions (0.5-8.0 mM) in the above buffer. 4MDM in 5% DMSO was added to each well at various concentrations (0-2.0 mM) and then 10 µg/ml LTA 4 H was added to the well with incubation at 30 °C for 10 min. Enzyme activity was measured by continuously monitoring the increase in absorbance at 405 nm for 30 min with 10 s intervals at 30 °C immediately following the addition of the substrate. Six replicates were run for each 4MDM and substrate combination and all readings were measured using a Bio-Tek Powerwave. PGP assays were performed at 30 °C in 1xPBS buffer pH 7.2 using a 96-well plate (Corning Costar). Proline-glycine-proline (PGP, Biomatik) was prepared at various concentrations (100-1000 μM) in the above buffer. 4-MDM dissolved in 5% DMSO were added to each well with various concentrations (0-0.32 μM) and then LTA4H (31.25 ng/ml) was added to 200 μL well for 30 min incubation at 30 °C. The enzymatic reactions were stopped at different time points, then the enzyme activity was measured using the fluorescamine derivatization method (Tecan Spark 10 M Spectrophotometer). The standard curve for Gly-Pro was determined, in advance, and used for enzyme activity data analysis.

Methods
Murine model of acute lung inflammation induced by LPS. All  BALF sample cleanup for PGP quantification using GC/MS. BALF samples were first filtered to remove salts and unwanted biological components present within the sample matrix. This removal was accomplished through the use of Waters C-18 Plus Short Cartridge Sep-Paks, with 360 mg of sorbent (Waters Coporation). Briefly, cartridges were conditioned by preforming three washes with acetonitrile (FisherScientific) followed by three washes with a 0.1% (v/v) trifluoroacetic acid (TFA) (Chem-Impex Internation) in MilliQ water solution, using a 1 mL Air-Tite All-Plastic Norm-Ject syringe (Air-Tite Products Co Inc) to dispense each solvent. BALF samples were then acidified with 1 μL of TFA, after which 300 μLs were drawn up and pushed through the cartridge at a rate of 1-3 drops per second. An additional two washes with the 0.1% (v/v) TFA in MilliQ water solution was then performed to remove any components in the sample matrix not compatible with the LC-MS analysis. Finally, bound PGP was eluted from the cartridge using a 20% (v/v) acetonitrile in MilliQ water solution. Eluate was dried in a SpeedVac, and resuspend in 300 μLs of a 0.1% (v/v) formic acid (FA) (Sigma Aldrich) in MilliQ water solution.

GC/MS instrumental analysis. PGP quantification was carried out on a TSQ Quantum Ultra Triple
Quadrupole mass spectrometer (ThermoFisher Scientific) connected to an Accela HPLC pump (ThermoFisher Scientific). Samples were injected onto a 1 mm × 150 mm Hypersil Gold 3 μm particle C-18 reversed phase column (ThermoFisher Scientific) using an Accela autosampler (ThermoFisher Scientific). Mobile phase A consisted of 0.1% FA in MilliQ water. Mobile phase B consisted of 0.1% FA in methanol. An isocratic gradient of 90% A and 10% B at a flow rate of 50 μLs min −1 was employed for the first 6 min of the run. Then, following the primary elution of PGP at 3.5-min mark, the gradient shifted to 100% B and held there for an additional minute before returning to initial conditions for the duration of the run, allowing for column regeneration. The mass spectrometer was run in the selected reaction monitoring (SRM) mode to detect PGP present in BALF samples. Transitions pairs used for SRM analysis have been provided in Table S3. Stock solutions of PGP were prepared from lyophilized PGP (Bachem, Bubendorf, Switzerland) in phosphate buffered saline (PBS) pH 7.4 (ThermoFisher Scientific) at six concentrations, ranging from 156 to 5000 pg mL −1 . Standards were prepared fresh and extracted the same day as BALF samples to ensure consistent processing.
Statistics. Prism v9.2.0 (Graphpad) was used for all statistical analyses. Murine study results were analyzed by two-way ANOVA with Bonferroni corrected subgroup analyses. P valued less than 0.05 was considered significant. Enzyme kinetic samples were prepared in six replicates (n = 6), and percent consumptions for each well was calculated and selected for the initial velocity using the pNA standard curve line of best fit. The coefficient of variation is within 5%.

Data availabilty
The data that support the findings of this study are available in the Supplementary Information file, and from the corresponding authors upon request. Coordinates and structure factors for all structures have been deposited to the Protein Data Banks, with the accession numbers of 7KZE and 7LLQ.